The phorbol esters are the most potent class of tumor promoters for mouse skin. Recently, this laboratory has demonstrated specific binding of the phorbol esters to chicken embryo fibroblasts, mouse skin, and mouse tissue preparations. This binding is saturable, competible, and reversible. Phorbol 12-myristate 13-acetate (PMA) has the highest binding affinity (7 x 10 to the minus 11th power M to 2 x 10 to the minus 9th power M, depending on the system); other phorbol esters have affinities corresponding to their tumor promoting activities. Highly irritant but non-promoting phorbol-related diterpenes bind weakly or not at all. The binding thus appears to be to the phorbol ester receptor involved in tumor promotion. It is proposed in this renewal to extend the analysis of the phorbol ester receptor. 1) Detailed characterization of the receptor will be carried out. Particularly emphasized will be variation in binding with cell and tissue type, growth parameters, transformation, and pharmacological agents; high resolution localization by autoradiography; and affinity labeling. 2) Genetic confirmation for the postulated role of the receptor will be sought. PMA-unresponsive variants of Friend erythroleukemia cells will be isolated to provide genetic evidence for the role of the receptor in biological responses. To confirm the role of the receptor in promotion, the relationship between receptor levels in mouse strains and their susceptibility to promotion will be examined. 3) Purification of the receptor by affinity and conventional techniques will be attempted. 4) Identification will be attempted of the biochemical response which is directly associated with binding of phorbol ester to its receptor, i.e. the second messenger, and which is in turn responsible for the biological cascade resulting from phorbol ester binding. 5) The receptor assay will be used to screen for potential endogenous compounds interacting at the same receptor.